Dandruff, alternatively referred to in the literature as ptyriasis simplex, furfuracea or capitis, is a skin disorder characterized by flaking, itching and microinflammation. By definition, dandruff is confined to the scalp, and it is experienced by about half of the adult population irrespective of ethnicity and gender. Dandruff has conventionally been considered trivial from a medical standpoint, but represents a persistent cosmetic concern.
Dandruff is considered to be a form of seborrheic dermatitis, which may be found in other locations on the body. Scalp scaling, which is present in both disorders, is not the clinical distinguishing feature. Rather, inflammation and presence of lesions outside the scalp exclude the diagnosis of dandruff. Generally, dandruff is considered the mildest form of seborrheic dermatitis from a clinical perspective since inflammation is minimal and typically subclinical. In fact, as recently as a decade ago the dandruff condition was thought to be non-inflammatory. The term “dandruff” is also used to describe the symptomatic scale itself, which has conventionally been considered as subject to cosmetic management. Response to currently available cosmetic treatments, however, is often transient. In contrast, seborrheic dermatitis is a more inflammatory disease and medical treatment is often undertaken to clear the disease, although cosmetics may also demonstrate some efficacy in reducing symptoms.
Dandruff scale comprises a cluster of corneocytes which have retained a large degree of cohesion with one another and which become detached as such from the surface of the stratum corneum. The size and degree of scaling is inconsistent across the region of the scalp and as a function of time. One hypothesis of dandruff formation relates to a suppression of the lipases responsible for proper shedding of corneocytes at the skin surface.
The pathogenesis of dandruff is complex, and appears to be the result of interactions between scalp skin, cutaneous microflora and the cutaneous immune system. The key clinical features of dandruff include flaking and itch, and although much descriptive work has been done, the precise underlying events that provoke these symptoms are incompletely understood. Dandruff is considered to have multiple sometimes overlapping causes with numerous pathogenic pathways and complex mechanisms. A microbial flora is implicated in the most common forms of dandruff. Generally, a healthy normal scalp is known to harbor many microbes reaching a density of 103 to 105 organisms per mm2 and including, for example, Staphylococci, Propionibacterium spp. and Malassezia spp.
The theory that dandruff is fundamentally a Malassezia-based disorder remains prevalent. However, as the clinical evidence implicating Malassezia in dandruff conditions and seborrheic dermatitis has accumulated over time, it has been observed that results obtained from quantitative methods used to count yeasts are inconsistent and do not correlate. For example, no relationship has been found between severity of symptoms and fungal count. It is hypothesized that only yeasts that are tightly bound to the skin correlate to the dandruff condition. Further perplexing however is that quantitative microbiological assessments fail to implicate a role specifically for yeast. Some investigators attribute this to a failure to note the relationship between dandruff and particular species of Malassezia. Regardless of the inability to account for the inconsistent empirical observations, controlling yeast of the genus Malassezia has heretofore been the most successful dandruff management strategy. Application of antifungal-based antidandruff shampoos generally results in lessening or disappearing itch after only a few applications. A problem with this strategy remains in that although anti-fungal management results in a decrease in the yeasts, complete eradication is rarely achieved. Scale production reduces in parallel to microbe reduction, yet within 2 to 3 weeks of ceasing treatment, dandruff recurs and Malassezia populations increase to their pre-treatment levels.
Close inspection reveals that the Malassezia yeasts appear in scattered clumps restricted in distribution over some corneocytes but not others. It has been hypothesized that dandruff represents a failure of a normal immune response by the specific keratinocytes where Malassezia yeasts are found. Other investigators note that Malassezia have antigenic and pro-inflammatory properties stimulating both the innate and acquired immune responses. Anti-inflammatory drugs such as dermocorticoids have proven efficacy, particularly in severe dandruff. Nonetheless, providing an anti-fungal active remains the conventional treatment of choice.
A further confounding problem in determining the causative basis of dandruff is the mode of action of the anti-fungal agents themselves. Most of the known actives such as zinc pyrithione (ZPT), a biocide widely recognized as an effective anti-fungal agent in shampoo formulations, are substantially insoluble in water so that sustained contact time with the scalp is very brief. Many investigators therefore posit that the anti-fungal agents exhibit efficacy through some ancillary mode of action including some direct biological effects on epidermal cells.
Scaling conditions similar to dandruff may occur with desquamation of the scalp following excessive exposure to sunlight where intercorneocyte cohesion is also affected, as well as in minor chronic irritation of the scalp. Further, over-brushing, over-shampooing, certain cosmetic hair products, and irritation from airborne substances may cause scaling. Other non-fungal causes include use of sebum-derived products, sunlight activation of follicular-photosensitizing agents such as porphyrins, and some neuro-immune conditions. Psychological stress is also widely considered to exacerbate dandruff.
Sebum has been found to be a prerequisite for dandruff, but not a sufficient factor per se. Many people who complain about oily scalp have no dandruff, while successful treatment of dandruff often leads to an increased coating of the hair shafts by sebum. Epidermal lipids exhibit differences across the dandruff condition both in quantity and quality. In particular, it has been demonstrated that the three main classes of stratum corneum lipids, i.e. ceramides, free fatty acids and cholesterol, display diminished content in dandruff-affected relative to healthy scalp skin.
Dandruff severity ranges from mild and discrete to severe and pervasive among affected individuals. Amount of scalp hair is a factor, although amounts of dandruff on the scalp and on hair are not always correlated. Products used to treat dandruff have been observed to suppress androgenic alopecia, and ketoconazole has been reported to stimulate hair growth in mice, among many other effects.
Dandruff, therefore, represents a reactive response of the epidermis of the scalp to various stimuli, some of which may be external and some of which may be internal, in combination with an individual predisposition, and its etiological complexity makes it a treatment challenge. There is a persistent need in the art for methods of identifying potential anti-dandruff agents and for evaluating the efficacy of putative agents having efficacy substantially independent of mechanism of action or etiology of the dandruff condition. The present investigators therefore undertook an investigation into the application of “connectivity mapping” to the search for new skin-active agents with efficacy in the treatment of dandruff and related skin conditions.
Connectivity mapping is a well-known hypothesis generating and testing tool having successful application in the fields of operations research, telecommunications, and more recently in pharmaceutical drug discovery. The undertaking and completion of the Human Genome Project, and the parallel development of very high throughput high-density DNA microarray technologies enabling rapid and simultaneous quantification of cellular mRNA expression levels, resulted in the generation of an enormous amount of gene expression data. At the same time, the search for new pharmaceutical actives via in silico methods such as molecular modeling and docking studies stimulated the generation of vast libraries of potential small molecule actives. The amount of information linking disease to gene expression profiles, gene expression profiles to drugs, and disease to drugs grew exponentially, and application of connectivity mapping as a hypothesis testing tool in the medicinal sciences ripened.
The general notion that functionality could be accurately determined for previously uncharacterized genes, and that potential targets of drug agents could be identified by mapping connections in a data base of gene expression profiles for drug-treated cells, was spearheaded in 2000 with publication of a seminal paper by T. R. Hughes et al. [“Functional discovery via a compendium of expression profiles” Cell 102, 109-126 (2000)], followed shortly thereafter with the launch of The Connectivity Map (—map Project by Justin Lamb and researchers at MIT (“Connectivity Map: Gene Expression Signatures to Connect Small Molecules, Genes, and Disease”, Science, Vol 313, 2006.) In 2006, Lamb's group began publishing a detailed synopsis of the mechanics of C-map construction and installments of the reference collection of gene expression profiles used to create the first generation C-map and the initiation of an on-going large scale community C-map project, which is available under the “supporting materials” hyperlink at http://www.sciencemag.org/content/313/5795/1929/suppl/DC1.
The basic paradigm of predicting novel relationships between disease, disease phenotype, and drugs employed to modify the disease phenotype, by comparison to known relationships has been practiced for centuries as an intuitive science by medical clinicians. Modern connectivity mapping, with its rigorous mathematical underpinnings and aided by modern computational power, has resulted in confirmed medical successes with identification of new agents for the treatment of various diseases including cancer. Nonetheless, certain limiting presumptions challenge application of C-map with respect to diseases of polygenic origin or syndromic conditions characterized by diverse and often apparently unrelated cellular phenotypic manifestations. According to Lamb, the challenge to constructing a useful C-map is in the selection of input reference data which permit generation of clinically salient and useful output upon query. For the drug-related C-map of Lamb, strong associations comprise the reference associations, and strong associations are the desired output identified as hits.
Noting the benefit of high-throughput, high density profiling platforms which permit automated amplification, labeling hybridization and scanning of 96 samples in parallel a day, Lamb nonetheless cautioned: “[e]ven this much firepower is insufficient to enable the analysis of every one of the estimated 200 different cell types exposed to every known perturbagen at every possible concentration for every possible duration . . . compromises are therefore required,” Lamb, J. (2007) “The Connectivity Map: a new tool for biomedical research” Nat. Rev. Cancer 7, 54-60, (page 54, column 3, last paragraph). Lamb, however, took the position that cell type did not ultimately matter, and confined his C-map to data from a very small number of established cell lines out of efficiency and standardization concerns. Theoretically this leads to heightened potential for in vitro to in vivo mismatch, and limits output information to the context of a particular cell line. If one accepts the Lamb precept that cell line does not matter then this limitation may be benign.
However, agents suitable as pharmaceutical agents and agents suitable as cosmetic agents are categorically distinct, with the former defining agents selected for specificity and which are intended to have measurable effects on structure and function of the body, while the latter are selected for effect on appearance and may not effect structure and function of the body to a measurable degree. Cosmetic agents tend to be non-specific with respect to effect on cellular phenotype, and administration to the body is generally limited to application on or close to the body surface.
In constructing C-maps relating to pharmaceutical agents, Lamb stresses that particular difficulty is encountered if reference connections are extremely sensitive and at the same time difficult to detect (weak), and Lamb adopted compromises aimed at minimizing numerous, diffuse associations. Since the regulatory scheme for drug products requires high degrees of specificity between a purported drug agent and disease state, and modulation of disease by impacting a single protein with a minimum of tangential associations is desired in development of pharmaceutical actives, the Lamb C-map is well-suited for screening for potential pharmaceutical agents despite the noted compromises.
The connectivity mapping protocols of Lamb would not be predicted, therefore, to have utility for hypothesis testing/generating in the field of cosmetics. Cosmetic formulators seek agents or compositions of agents capable of modulating multiple targets and having effects across complex phenotypes and conditions. Further, the phenotypic impact of a cosmetic agent must be relatively low by definition, so that the agent avoids being subject to the regulatory scheme for pharmaceutical actives. Nonetheless, the impact must be perceptible to the consumer and preferably empirically confirmable by scientific methods. Gene transcription/expression profiles for cosmetic conditions are generally diffuse, comprising many genes with low to moderate fold differentials. Cosmetic agents, therefore, provide more diverse and less acute effects on cellular phenotype and generate the sort of associations expressly taught by Lamb as unsuitable for generating connectivity maps useful for confident hypothesis testing.
Nonetheless, contrary to the teachings of Lamb and the prior art in general, the present inventors surprisingly discovered that useful connectivity maps could be developed from cosmetic active—cellular phenotype—gene expression data associations in particular with respect to skin-care actives and cosmetic agents, despite the highly diffuse, systemic and low-level effects these sorts of actives generally engender. Further, contrary to assertions by the Lamb team that results should be substantially independent of cell-type, the present invention is based in part on the surprising discovery that selection of human epidermal keratinocyte cells as the relevant cell line resulted in construction of connectivity maps particularly useful for hypothesis generating and testing relating to skin-active agents and cosmetic agents useful in the treatment of dandruff.
As noted above, the dandruff condition is particularly complex and its etiology is not fully understood. The present investigators therefore made a novel adaptation to the C-map paradigm that has proven to be particularly useful in identifying agents with potential efficacy in certain skin diseases, including dandruff. Although gene expression signatures are determined for the skin condition, the gene expression signature is further analyzed to determine an implicated biological process pattern which is used to derive a physiological thematic expression signature. The theme signature is then used to query the C-map data base to generate a skin-active agent output where highly negative connectivity to the skin condition thematic expression signature predicts efficacy for treatment of the skin condition. To the best knowledge of the present investigators, application of connectivity mapping to target a multi-factored, poorly delineated and low-level “disease” condition such as dandruff, by identifying agents through the use of physiological theme expression signatures has not been attempted previously.
The present investigators further discovered that a well-designed connectivity map may provide insights into the pathogenesis of the skin condition and the mechanism of action of benchmark actives. By application of C-map, the present inventors surprisingly discovered, for example, modes of action for anti-fungal agents that are independent of anti-fungal properties. Further, by conducting the transcriptional profiling analyses as part of the C-map process, the present inventors surprisingly discovered that by inspecting a gene expression signature for biological process themes, in vitro models of disease states could be constructed with a surprisingly high fidelity to the clinical disease state with respect to response of the gene expression profile to specific skin-active agents.
Successful identification of anti-dandruff agents has proven to be difficult due to the multi-cellular, multi-factorial processes involved in etiology of the dandruff condition itself. Conventional in vitro studies of biological responses to potential anti-dandruff agents can be hindered by the complex or weakly detectable responses typically induced and/or caused by the putative or potential agents. Such weak responses arise, in part, due to the great number of genes and gene products involved, and skin-active and cosmetic agents may affect multiple genes in multiple ways. Moreover, the degree of bioactivity of cosmetic agents may differ for each gene and be difficult to quantify.
The value of a connectivity map approach to discover functional connections among cosmetic phenotypes such as aged skin, gene expression perturbation, and cosmetic agent action is counter-indicated by the progenitors of the drug-based C-map. The relevant phenotypes are very complex, the genetic perturbations are numerous and weak, and cosmetic agent action is likewise diffuse and by definition, relatively weak. It was considered unlikely that statistically valid data could be generated from cosmetic C-maps and it was unclear whether a cell line existed which could provide salient or detectable cosmetic data.